Project description:Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however, the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms.

| S-EPMC4272677 | biostudies-literature

Thermophilic phosphoribosyltransferases <i>Thermus thermophilus</i> HB27 in nucleotide synthesis.

Project description:Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(?-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-?l-AMP) using TthAPRT and 1-(?-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using Tth?PRT.

| S-EPMC6317416 | biostudies-literature